1. Field of the Invention
This invention relates to the guided transport of biological molecules or cells to which small magnetic particles have been attached, particularly when such molecules or cells are then to be detected optically in a chemical or biological assay.
2. Description of the Related Art
Physical extraction of biological cells and molecules from liquid biological solutions by exerting magnetic forces on attached magnetic labels (i.e., small magnetized particles) has been a widely adopted technique in medical and biological practice. The biological cells or molecules have magnetic labels attached to them, the labels being very small particles of magnetic material that are magnetizable by an external magnetic field. Such small particles of magnetic material are typically superparamagnetic, meaning that thermal effects are sufficiently large to destroy spontaneous domain formation and, therefore, they must be placed in an external magnetic field to acquire a magnetization. Thus, detection of the target cells or molecules is usually accomplished by applying such an external magnetic field that magnetizes the magnetic labels, exerts a magnetic force on them and extracts them from the liquid-form samples together with the cell and molecule to which they attach. Afterwards, a subsequent reading of, for example, optical signals emitted by fluorescent or luminescent compounds (dyes) previously also attached to the extracted cells or molecules is performed to identify the existence of the target molecules or cells. However, such an ensemble oriented extraction technique is incapable of producing detections at the single molecule level, because the target molecules are detected in the form of concentrated clusters or as droplets where signal scattering by unbound labels or liquid solution can be very high.
Referring to FIGS. 1A-1D, there is shown a schematic illustration of such a prior art method of magnetically extracting and optically detecting magnetically labeled molecules. In FIG. 1A there is seen a biological solution (1) containing the target molecules (2) to be detected and distinguished from molecules that are not of interest (3). FIG. 1B shows the target molecules (2) with magnetic labels (4) and fluorescent dyes (5) attached to them. FIG. 1C shows the fluid (1), passing in a channel (8) between the poles of a magnet (7). Solid arrows indicate the magnetization of the magnet. The magnetic labels have been attracted to either side of the channel by the interaction between the external magnetic field of the magnet and the induced magnetization within the labels, pulling their attached molecules with them. In FIG. 1D there is shown a subsequent identification of the labeled target molecules (4) by means of a beam of excitation light (9) and the optical detection of excitation fluorescence (10) in an optical detection system. (11). M. A. Reeve, (U.S. Pat. No. 5,523,231) teaches a method to isolate macromolecules using such magnetically attractable particles. Similarly, M. A. M. Gijs has published “A Magnetic bead handling on-chip: new opportunities for analytical applications,” Microfluid Nanofluid. Pp 22-40, 2004.
The prior art also teaches detection of labeled biological molecules or viruses with accuracy at the level of single molecules by the use of magneto-resistive (MR) sensors. D. R. Baselt et al., “A biosensor based on magnetoresistance technology,” Biosens. Bioelectron., vol. 13, pp. 731-739, October 1998, M. M. Miller et al., “A DNA array sensor utilizing magnetic microbeads and magnetoelectronic detection,” J. Magn. Magn. Mater., vol. 225, pp. 138-144, April 2001 and S. X. Wang et al., “Towards a magnetic microarray for sensitive diagnostics,” J. Magn. Magn. Mater., vol. 293, pp. 731-736, 2005.
Referring to FIG. 2, there is schematically shown how such a prior art system can operate. The technique usually uses a regular array of identical MR sensors, one such sensor being indicated as (12). Each sensor is formed between an intersection of two sets of vertically separated horizontally directed parallel current carrying wires (160), (16) that are orthogonal to each other.
The individual patterned magnetic devices comprise two horizontal electrically conducting planar magnetic layers (13), (14), separated by a non-magnetic layer (15) and the array may be formed by patterning a larger horizontal film deposition of two horizontal planar magnetic layers separated by a non-magnetic layer.
Subsequent to (or prior to) their being patterned into the array of discrete devices (12), the magnetic layers are magnetized and the magnetization of one of the layers (nominally, the “bottom” layer (14)) is fixed in spatial position and may be denoted the “pinned” layer and the magnetization of the other layer (nominally the “top” layer (13)) is allowed to move freely and may be denoted the “free” layer. The direction of the magnetization of each layer is predisposed by providing the layers with some form of magnetic anisotropy, either a crystalline anisotropy that results from the layer deposition process or a shape anisotropy that results from the patterning, or both.
As a result, by a proper choice of currents in the two sets of wires (100), (16), the magnetization of the free layer can be moved and can be caused to be parallel to or anti-parallel to that of the fixed layer. It is well known in the prior art that such sensors display two resistance states according to the relative directions of the two magnetic moments. When the moments are aligned (parallel), the resistance is low and when the moments are anti-aligned (anti-parallel) the resistance is high. Thus, a measurement of the resistance of any element in the array will give an immediate indication of the alignment of its magnetizations. The basic idea is then to magnetize the label of the captured molecule (4) and to have its magnetization switch the direction of the free layer magnetization of the sensor element over which it is trapped. The switching is detected as a resistance change and it gives an indication of a trapped particle.
Typically such an array of sensors is formed beneath a substrate surface (not shown) that is furnished with chemical binding sites that are specific to the molecule or cell being detected. For simplicity of the figure and ease of visualization, a captured target molecule (2) and its attached magnetic label (4) is shown as being bound to one of the conducting lines (160). In practice, the conducting line is beneath the substrate and the molecule is bound to a site on the substrate surface. When such a molecule binds to one of the sites, its label is then in a fixed position over the portion of the sensor array beneath the binding site. In this figure, the molecule (2) is shown as being directly over one of the sensors (12). After the magnetic labels that are not bound to the substrate surface are removed, typically by flushing the surface, the remaining magnetic labels are subjected to an external magnetic field that is perpendicular to the substrate plane, whereupon the labels generate an induced magnetic field (17) that projects into the underlying MR sensor and is parallel to the magnetic layers of the sensor. As already noted above, because the magnetic particles are so small, they are “superparamagnetic”, meaning that thermal energy exceeds the energies that would create stable domains, so there is no spontaneous magnetization. Consequently, the particle must be subjected to an external magnetic field so that it may become magnetized and produce its own magnetic field. The surface attachment of the magnetic labels ensures their close proximity to adjacent MR sensors, to enhance the effects of the small magnetic signal they generate. However, this method does require the process of capturing the target molecules on the substrate surface, as well as the removal of the labels that do not have their molecules attached to surface sites. Since label binding to molecule and molecule binding to surface requires two separate incubation processes, this new method is theoretically slower than the conventional optical method in its preparation step, because in the optical identification method a single incubation is enough to accomplish both magnetic label attachment and dye attachment to the target molecules. In addition, the MR signal variation between patterned MR matrix cells can be sufficiently great so that the magnetic labels need to exceed a certain size to achieve acceptable accuracy and repeatability in their detection.
We will note at this point that studies within the prior art have shown that sensor arrays such as those illustrated in FIG. 2, can also be used to move small magnetic particles, rather than to detect them. It was shown in prior arts by E. Mirowski et al., “Manipulation of magnetic particles by patterned arrays of magnetic spin-valve traps,” J. Magn. Magn. Mat., vol. 311, pp. 401-404, 2007 and by J. Moreland et al., “Microfluidic platform of arrayed switchable spin-valve elements for high throughput sorting and manipulation of magnetic particles and biomolecules,” Moreland, also in US published patent application 2005/0170418, teaches that physical manipulation of a single magnetic particle can be achieved with patterned arrays of magnetic multi-layer thin film structures. The magnetic particles can be trapped by a magnetic pattern and later released from the pattern by switching the magnetization of one of the magnetic layers between different directions. Referring to FIGS. 3A and 3B, there is shown schematically how such a prior art process achieves these objects by an illustration with a single labeled particle and a single trilayered device formed by patterning a multilayered thin film structure.
The patterned device (12) in both FIGS. 3A and 3B includes a free magnetic layer (13) formed of a magnetic material such as CoFe, a non-magnetic inter-layer (15), formed of a dielectric material such as AlOx and a pinned layer (14), formed of material similar to that of the free layer. A single molecule (2) to which is attached a magnetic label (4) is adjacent to the device in FIG. 3A. A switching current (19) Iswitch in an adjacent electrical line (16) rotates the magnetic moment (50), Mfree of the free layer so that it is parallel to the magnetic moment (6), Mpinned, of the pinned layer. The parallel magnetic moments effectively produce magnetic charges on the lateral edges of the device which, in turn, induces a magnetization (7), Mlabel, in the label (4). The induction process produces a net magnetic attraction between the lateral edge of the device and the magnetized label, bringing the label to the device and trapping it there.
Referring next to FIG. 3B, there is shown schematically the configuration of FIG. 3A wherein the switching current (19). Iswitch in the line (16) has been reversed in direction, causing the magnetic moment (5) of the free layer (13) to reverse direction and become antiparallel to the magnetic moment (6) of the pinned layer (14). The lateral edges of the device now have net zero magnetic charge, releasing the label (4) and allowing its induced magnetization to essentially disappear.
In whatever method of detection is used, in order to achieve a speedy detection and counting process at the single molecule level, it is preferable that the biological preparation steps be as simple as possible. For example, the one-step incubation process, as used in the conventional optical method described in FIGS. 1A-1D is regarded as being advantageous compared with the MR assay method as described in FIG. 2.
However, to realize single molecule counting, the biological cells or molecules must be manipulated and detected individually, producing sufficient physical separation to ensure the separate response of each individual molecule in space or in time. This is a basic requirement. The conventional ensemble magnetic label extraction and optical detection scheme illustrated in FIG. 1A-1D will not be able to separate each individual label or molecule, even using state-of-the-art flow-cytometry or micro-fluidics systems. In short, the MR sensing scheme illustrated in FIG. 2 is more likely to accomplish the goal of single molecule detection due to the controllable spatial separation between individual captured molecules that it provides.
U.S. Patent Application 2005/0170418 (Moreland et al) discloses using spin valve elements to trap, hold, manipulate, and release magnetically tagged particles, but there is no disclosure of transporting the particles. The prior art also discloses the following patents. U.S. Pat. No. 5,523,231 (Reeve) teaches magnetic extraction of molecules using magnetic beads. U.S. Pat. No. 5,691,208 (Miltenyi et al) shows magnetic spheres in a lattice format used to separate labeled cells from a fluid. U.S. Pat. No. 6,294,342 (Rohr et al) shows an assay method of binding magnetically labeled particles. U.S. Pat. No. 7,056,657 (Terstappen et al) teaches trapping and releasing magnetically labeled cells, but there is no disclosure of transport.
As noted above, each of the prior art methods, including optical detection and MR sensor detection, has its advantages and disadvantages. None of them provide a robust method of reliably detecting the presence of individual beads. It is the object of the present invention to provide such a method.